We measured soil CO2 flux over 19 sampling periods that spanned two growing seasons in a grassland Free Air Carbon dioxide Enrichment (FACE) experiment that factorially manipulated three major anthropogenic global changes: atmospheric carbon dioxide (CO2) concentration, nitrogen (N) supply, and plant species richness. On average, over two growing seasons, elevated atmospheric CO2 and N fertilization increased soil CO2 flux by 0.57 µmol m−2 s−1 (13% increase) and 0.37 µmol m−2 s−1 (8% increase) above average control soil CO2 flux, respectively. Decreases in planted diversity from 16 to 9, 4 and 1 species decreased soil CO2 flux by 0.23, 0.41 and 1.09 µmol m−2 s−1 (5%, 8% and 21% decreases), respectively. There were no statistically significant pairwise interactions among the three treatments. During 19 sampling periods that spanned two growing seasons, elevated atmospheric CO2 increased soil CO2 flux most when soil moisture was low and soils were warm. Effects on soil CO2 flux due to fertilization with N and decreases in diversity were greatest at the times of the year when soils were warm, although there were no significant correlations between these effects and soil moisture. Of the treatments, only the N and diversity treatments were correlated over time; neither were correlated with the CO2 effect. Models of soil CO2 flux will need to incorporate ecosystem CO2 and N availability, as well as ecosystem plant diversity, and incorporate different environmental factors when determining the magnitude of the CO2, N and diversity effects on soil CO2 flux.